Homogeneous MMR Deficiency Throughout the Entire Tumor MassOccurs in a Subset of Colorectal Neuroendocrine Carcinomas

Christoph Fraune1 & Ronald Simon1& Claudia Hube-Magg1

& Georgia Makrypidi-Fraune1 & Martina Kluth1&

Franziska Büscheck1 & Tania Amin3& Fabrice Viol3 & Wilfrid Fehrle1 & David Dum1

& Doris Höflmayer1 & Eike Burandt1 &

Till Sebastian Clauditz1 & Daniel Perez2 & Jakob Izbicki2 & Waldemar Wilczak1 & Guido Sauter1 & Stefan Steurer1 &

Jörg Schrader2,3

Published online: 6 March 2020# The Author(s) 2020

AbstractNeuroendocrine neoplasms comprise a heterogeneous group of tumors, categorized into neuroendocrine tumors (NETs) andneuroendocrine carcinomas (NECs) depending on tumor differentiation. NECs and high-grade NETs (G3) confer a poor prog-nosis, demanding novel treatment strategies such as immune checkpoint inhibition in tumors with microsatellite instability(MSI). To study any possible intratumoral heterogeneity of MSI, a tissue microarray (TMA) containing 199 NETs and 40NECs was constructed to screen for MSI using immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1,PMS2, MSH2, and MSH6. Four cases suspicious for MSI were identified. Validation of MSI by repeated IHC on large sectionsand polymerase chain reaction (PCR)–based analysis using the “Bethesda Panel” confirmed MSI in 3 cecal NECs. One pancre-atic NET G3 with MSI-compatible TMA results was MMR intact on large section IHC and microsatellite stable (MSS). Theremaining 235 tumors exhibited intact MMR. Protein loss of MLH1/PMS2 was found in two and MSH6 loss in one cancer withMSI. Large section IHC on all available tumor-containing tissue blocks in NECs with MSI did not identify aberrant tumor areaswith intactMMR. Our data indicate thatMSI is common in colorectal NECs (3 out of 10) but highly infrequent in neuroendocrineneoplasms frommany other sites. The lack of intratumoral heterogeneity ofMMR deficiency suggests early development ofMSIduring tumorigenesis in a subset of colorectal NECs and indicates that microsatellite status obtained from small biopsies may berepresentative for the entire cancer mass.

Keywords Microsatellite instability . Tissue microarray . Neuroendocrine tumor . Neuroendocrine carcinoma

Introduction

Neuroendocrine neoplasms occur at various organ sites andcomprise a heterogeneous group of epithelial tumors rangingfrom well-differentiated neuroendocrine tumors (NETs) topoorly differentiated carcinomas, called neuroendocrine

carcinomas (NECs). NETs clinically often present as low-grade malignancies, while NECs behave more aggressivelywith rapid disease progression and poor long-term survival,demanding for novel treatment options.

Microsatellite instability (MSI) refers to a distinctive pat-tern of increased mutational load in certain tumors, typicallycaused by a defective DNA mismatch repair (MMR) appara-tus, and is characterized by accumulated length variations ofrepetitive DNA sequences (microsatellites) throughout the ge-nome. Protein loss of at least one of the major components ofthe MMR system—MLH1, PMS2, MSH2, or MSH6, whichcan easily be analyzed by immunohistochemistry (IHC)—provides strong indirect evidence for MSI. MSI can also bedetected via direct analysis of a predefined panel of DNAmicrosatellite loci with classic polymerase chain reaction(PCR)–based methods or novel high-throughput moleculartechniques [1]. Favorable response rates for immune check-point inhibitors in cancers with MSI have dramatically

* Ronald SimonR.Simon@uke.de

1 Institute of Pathology, University Medical CenterHamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany

2 General, Visceral and Thoracic Surgery Department and Clinic,University Medical Center Hamburg-Eppendorf,Hamburg, Germany

3 I. Medical Department – Gastroenterology and Hepatology,University Medical Center Hamburg-Eppendorf,Hamburg, Germany

Endocrine Pathology (2020) 31:182–189https://doi.org/10.1007/s12022-020-09612-7

increased the clinical request for MSI testing, even in tumortypes with low expected rates of MSI. This was paralleled bythe 2017 site-agnostic FDA approval of the PD-1 antibodypembrolizumab for advanced cancers with MMR deficiency/MSI-high.

Data on MSI in neuroendocrine neoplasms are limited.Recent studies have found tumors with MSI among neuroen-docrine carcinomas (some combined with a conventional ad-enocarcinoma component, MINEN) of the stomach, small in-testine, and colorectum [2–6]. The few available studies de-scribing MSI in cohorts of NETs are basically limited to pan-creatic primaries with reported frequencies between 10%(5/48) and 33% (18/55) by PCR-based methods [7, 8].Intratumoral heterogeneity of MSI—a potential strong con-founder for molecular based treatments—has been observedin several cancer types, including colorectal cancer [9–13].However, intratumoral heterogeneity of MSI in neuroendo-crine neoplasms has not been evaluated so far.

To systematically assess potential intratumoral heterogene-ity of MMR protein expression in neuroendocrine neoplasmswith MMR deficiency, a cohort of 239 NETs and NECs wasscreened on a tissue microarray (TMA) format by IHC,followed by a large section evaluation of cancers suspectedfor MSI by repeated IHC and PCR analysis.

Material and Methods

Tissue Microarray A TMA comprising 239 neuroendocrineneoplasms and various control tissues was constructed usingone tissue core per tumor from archivedmaterial from patientsdiagnosed with a NET or NEC at the University MedicalCenter Hamburg-Eppendorf between 2009 and 2018. TMAconstruction was described earlier [14]. Clinical and patholog-ical information were obtained from the patient’s medical re-cords and reports. NET- and NEC-categorization was basedon the criteria described in the recent 5th edition of the WHOclassification of tumors of the digestive system, and differen-tiation between NECs and high-grade (G3) NETs was adoptedfrom Tang et al. [15]. No mixed tumors with an additionalnon-neuroendocrine tumor component (MINEN) were identi-fied. Utilization of archived diagnostic leftover tissues formanufacturing of TMAs and their analysis for research pur-poses as well as patient data analysis has been approved bylocal laws (HmbKHG, §12,1) and by the local ethics commit-tee (Ethics commission Hamburg, WF-049/09). All work hasbeen carried out in compliance with the Helsinki Declaration.

Immunohistochemical Analyses To screen for MSI cancers,freshly taken TMA sections were immunostained on 1 dayand in one experiment using an automated immunostainer(Dako/Agilent Autostainer Link 48). Primary antibody specif-ic for MLH1 (clone ES05, mouse), PMS2 (clone EP51,

rabbit), MSH2 (clone FE11, mouse), and MSH6 (cloneEP49, rabbit) (all Ready-to-Use, all from DAKO, Glostrup,Denmark) was applied for 20 min (MLH1, MSH2, MSH6) or30 min (PMS2). Bound antibody was visualized using theEnVision Kit (Dako, Glostrup, Denmark) according to themanufacturer’s directions. Tumors were scored as negative(0) when nuclear staining was absent in all captured tumorcells and positive (+) when unequivocal nuclear staining intumor cells (irrespective of staining intensity or percentage ofstained cells) was observed. In spots showing a negative (0)result for the tumor cells, presence (+) or absence (−) of nu-clear staining in peritumoral stromal or inflammatory cellswas also recorded (internal control). For TMA spots with astaining loss of any MMR protein in tumor cells but not instromal/inflammatory cells (suspected MSI), IHC was repeat-ed on a corresponding large section of the routinely archivedtumor material and PCR analysis was performed. In case ofnon-interpretable TMA spots (lack of unequivocal tumor tis-sue or negative (0) tumor cells accompanied by absence of aninternal control (−)), staining was repeated on a representativelarge section.

PCR Analysis For all cases with suspected MSI based on TMAscreening, a fluorescent PCR-based assay was performed(MSI Analysis System; Promega, Madison, WI), incorporat-ing the five microsatellite loci of the “Bethesda Panel” whichincludes two mononucleotide repeats (BAT25, BAT26) andthree dinucleotides repeats (D2S123, D5S346, andD17S250) [16]. Analysis was based on DNA extracted fromtumor tissue that was dissected from a large section of therespective tumor block corresponding to the TMA case andfrom non-neoplastic control tissue of the patient. Percentageof tumor cells was at least 50%within the analyzed tissue area.Status of MSI-high was given when at least 2 (≥ 40%) of themarkers showed instability (i.e., length variation comparedwith control tissue) and status of MSI-low if one of the ana-lyzed loci showed instability; otherwise, microsatellite stabil-ity (MSS) was assumed.

Evaluation of Cancer Heterogeneity If MSI was confirmed, allavailable archived tumor-containing tissue blocks were ana-lyzed to search for intratumoral heterogeneity. All together 20large sections were analyzed for 3 patients with confirmedMMR deficiency.

Results

TMA Screening and Large Section Validation From 239 neu-roendocrine neoplasms, including 199 NETs and 40 NECs ofgastroenteropancreatic and pulmonary sites (Table 1), TMAscreening obtained interpretable results in 224 (94%) cases.Tumors were considered interpretable if either unequivocal

Endocr Pathol (2020) 31:182–189 183

loss of staining for at least one of the four MMR proteinsMLH1, PMS2, MSH2, or MSH6 with an internal positivecontrol (MMR deficient) or unequivocally retained expressionfor all 4 MMR proteins (MMR intact) was observed. For non-interpretable TMA cases—due to lack of unequivocal tumortissue on the TMA spot (n = 15)—large section IHC was per-formed and revealed intact MMR in each cases. Two hundredtwenty from the 224 tumors with interpretable results fromTMA screening were also MMR intact, resulting in 235 of239 neuroendocrine neoplasms demonstrating retained ex-pression of all four MMR proteins. The remaining 4 tumorswere all high-grade neuroendocrine neoplasms, including onepancreatic NET G3 and 3 colorectal NECs, demonstratingloss of one or two MMR proteins on the TMA accompaniedby an adequate internal positive control, and were thus con-sidered suspicious for MSI (Table 2). Representative micro-graphs from the three cecal NECs with MMR deficiency areshown in Fig. 1.

MSI Validation Large section validation confirmed MMR de-ficiency in 3 of 4 suspected cases, all representing NECs of thecolorectum (Table 2). Two of these cancers were negative forMLH1/PMS2 and one showed isolated protein loss of MSH6.All three were MSI-high by PCR-analysis, were located in thececum, and demonstrated large cell phenotype. No non-neu roendoc r i n e t umo r componen t wa s f ound .Clinicopathological data did not reveal any further clear-cutassociations as compared with colorectal NECs with intact

MMR (Table 3). The one tumor with suspected MSI basedon TMA screening that turned out MMR intact on large sec-tion analysis was a pancreatic NET G3 with strongly attenu-ated PMS2 immunoreactivity throughout the majority of thetumor, but PMS2 expression was still retained and the otheranalyzed MMR proteins were inconspicuous. A comparisonwith the PMS2 staining on a corresponding large section re-vealed that the TMA spot had unluckily been taken from anarea with markedly diminished immunoreactivity (Fig. 2).PCR analysis did not show any instability among the analyzedmicrosatellite loci (MSS) in this tumor.

MSI Heterogeneity Analysis In the 3 NECs with confirmedMSI, IHC for MLH1, PMS2, MSH2, and MSH6 from allavailable archived cancer-containing tumor blocks (n=20) re-vealed consistent MMR protein loss throughout the entire tu-mor mass. No concurrent tumor areas with intact MMR werefound.

Discussion

ATMA containing 199 NETs and 40 NECs of various prima-ry sites was manufactured for the purpose of this study as ascreening tool to identify tumors with MSI among the hetero-geneous group of neuroendocrine neoplasms. Detection ofrare events is an ideal application for TMAs. For example,earlier studies identified 42 tumors harboring IDH1 mutationsby screening 15,531 prostate cancers (0.3%) or 43 tumorsexhibiting CD117 overexpression in a cohort of 1654 breastcarcinomas (2.6%) [17, 18]. We recently also identifiedMMRdeficiency/MSI in 5 tumors in a series of 448 bladder cancers(1.1%), in 7 tumors in a series of 200 advanced and/or hor-mone refractory prostate cancers (3.5%), and in 9 tumors in aseries of 479 ovarian cancers (1.8%) (unpublished data). In thepresent study, only one of the 4 tumors with suspected MMRdeficiency/MSI based on TMA screening turned out to beMMR intact on subsequent large section analysis. The dis-crepant results between TMA and large section IHC wereobserved in a pancreatic NET G3 and attributable to subopti-mal immunoreactivity of major tumor areas, which—forPMS2—resulted in still faintly detectable staining of stromalcells but negativity of tumor cells on the TMA spot in contrastto unequivocally retained MMR protein expression in cancercells on large section. This observation may reflect somewhathigher PMS2 expression levels in a subset of stromal cells ascompared with cancer cells.

Among the 239 analyzed neuroendocrine neoplasms, MSIwas confirmed in 3 cases (1.3%), all representing NECs of thececum, suggesting a considerable prevalence of MSI (30%;3/10) in colorectal NECs, although this may not reflect thetrue MSI rate in colorectal NECs due to the small tumor co-hort. None of the other primary NECs from different sites of

Table 1 Study cohort of neuroendocrine neoplasms separated byanatomic site, tumor type and tumor grade

Site NET NEC Sum

G1 G2 G3 SC LC

Esophagus 1 4 5

Stomach 5 2 1 8

Duodenum 2 2 4

Ampulla Vateri 1 1

Jejunum 2 1 3

Meckel’s diverticulum 1 1 2

Ileum 36 17 1 1 55

Appendix 20 2 22

Colorectum 8 1 5 5 19

Pancreas 28 26 5 1 4 64

Gall bladder 1 3 1 5

Lung 12 7 2 5 26

Metastatic* 6 10 2 5 2 25

Sum 121 70 8 17 23 239199 40

*Primary tumor sites include ileum (7), pancreas (2), lung (2), rectum (1),and unknown (CUP; 6) for metastatic NETs and lung (2), appendix (1),and unknown (CUP; 4) for metastatic NECs

184 Endocr Pathol (2020) 31:182–189

the gastrointestinal tract (n = 15) and the lung (n = 7) exhibitedMSI. These data fit with previous studies reporting particular-ly high rates ofMSI in NECs of the colorectum. Sahnane et al.found MSI in 6 of 37 (16%), Olevian et al. in 2 of 29 (7%),Furlan et al. in 2 of 21 (10%), and La Rosa et al. in 3 of 35(14%) colorectal NECs [2, 4–6]. In the study by Sahnane et al.also 4 tumors with MSI were identified among 36 gastricNECs (11%) [2]. A high frequency of MSI (44%) based onIHC detection of MMR protein loss was further observed byPocrnich et al. in a cohort of 18 large cell NECs of the endo-metrium [19]. In contrast, NECs from other organs have rarely

been found to exhibit MSI. Sahnane et al. found only oneother tumor with MSI among 16 NECs of the duodenum,esophagus, pancreas, and gall bladder [2].

Taken together, the available data suggest that the preva-lence of MSI in NECs is site-dependent and closely related tothose organ sites where the exocrine neoplastic counterparts ofNECs—adenocarcinomas—are also frequently affected byMSI, such as endometrial, colorectal, and gastric adenocarci-nomas. All 3 colorectal NECs with MSI in the present studywere located in the cecum, corresponding well to the strongassociation of MSI in sporadic colorectal adenocarcinomas

Fig. 1 TMA spots of the three colorectal NEC with MSI. Two cancers showed loss of MLH1/PMS2 and one cancer isolated loss of MSH6

Table 2 Summary of IHC and PCR data on neuroendocrine neoplasms with suspected MSI based on TMA screening. Not all of the 5 microsatelliteloci of the “Bethesda Panel” were evaluable by PCR for each tumor with MSI; however, status of MSI was still unequivocal in each case

Tumor type TMA screening Large section validation Entire tumor

MLH1 PMS2 MSH2 MSH6 MLH1 PMS2 MSH2 MSH6 Status IHC PCR Tumor blocks(n =)

MMR pattern

PancreaticNET G3

+ − + + + +(w-eak)

+ + MMRintact

MSS (0/5) − −

ColorectalNEC

− − + + − − + + MMRdeficient

MSI-high(4/4)

5 Homogeneous MMRdeficiency

ColorectalNEC

+ + + − + + + − MMRdeficient

MSI-high(3/5)

7 Homogeneous MMRdeficiency

ColorectalNEC

− − + + − − + + MMRdeficient

MSI-high(3/3)

8 Homogeneous MMRdeficiency

Endocr Pathol (2020) 31:182–189 185

with tumor location in the right colon [20]. A link betweenMSI in conventional and neuroendocrine carcinomas of dis-tinct organ sites is further supported by several reports of MSIin NECs with concomitant adenocarcinoma components inthe affected tumors (MINENs) [2, 4, 6]. The concept of sitedependency of MSI in NECs is also supported by the reportedabsence of MSI-high tumors among 107 small cell lung can-cers by Chung et al. [21], paralleling the low frequency ofMMR deficiency in non-small cell lung cancer [22–26].Based on the available data, MSI should be regularly testedin colorectal, gastric, and endometrial NECs. As NECs confera very poor prognosis and no second line treatment after fail-ure of Cis/Carboplatin and Etoposide has been established[27], MSI testing should be performed during initial diagnosisto avoid unnecessary treatment delay upon progression afterfirst-line treatment. As colorectal NECs have less than 50%response rate upon Platin-based chemotherapy and an overallsurvival of less than 9 months, this patient group might inparticular benefit from upfront MSI testing and initiation ofimmune checkpoint inhibitor therapy [27].

In contrast to NECs, none of the 199 NETs exhibited MSIin the present study. ThatMSI is highly infrequent in NETs fitswell with data from earlier studies. MSI was not found inprevious studies analyzing 56 rectal [28], 14 small bowel[29], 16 pancreatic [30], 29 gastroenteropancreatic [31], and38 foregut/midgut [32] as well as 35 pancreatic and 34 smallintestinal NETs [33] by PCR. In contrast, two earlier studiesdescribed relevant MSI rates in pancreatic NETs [7, 8].However, as those studies were performed 10 and more yearsago, the applied approach to identify or define MSI differsfrom current standards. House et al. reported MSI in 5 of 48(10%) pancreatic NETs using the “Bethesda Panel,” but statusof MSI was already given when merely the mononucleotiderepeat locus BAT-25 was instable, contrasting the current cut-off requiring instability in 40% of loci to justify MSI [7]. Meiet al. reportedMSI-high, defined as instability of at least 4 lociof an extended panel of 12 microsatellites, in 18 of 55 (33%)insulinomas [8]. In contrast, all 8 insulinomas among the co-hort of 56 pancreatic NETs analyzed in the present study wereMMR intact. There are, however, reports describing MMRdeficiency in few individual cases of pancreatic NETs in pa-tients with hereditary MMR deficiency (Lynch Syndrome)[34, 35].

Evaluation of the MMR status throughout all availablecancer-containing tumor blocks revealed homogeneousMMR protein loss in all three NECs with confirmed MSI.This is in agreement with our previous observations of highhomogeneity of MSI in prostate [36], ovarian [37], and blad-der cancer (unpublished data). Overall, these data may suggestthat MMR inactivation generally occurs early in tumorigene-sis. Regarding treatment purposes, homogeneity of MSI re-duces the risk that molecular parameters obtained from smallbiopsies may not be representative for the entire cancer mass,Ta

ble3

MSI

status

andclinicopathologicalp

aram

etersin

colorectalNECs

No.

MMRdeficiency

Location

Age

Gender

Smallcell/large

cell

Mito

ses(10HPF

)Ki-67

(%)

CD56

Syn

Chrom

oMIN

EN

TNM

1Yes

Cecum

95f

LC

5750

+++,100%

++,100%

−−

pT4,pN

0

2Yes

Cecum

59m

LC

3940

++,100%

+++,100%

+++,100%

−pT

2,pN

0,pM

1(H

EP)

3No

Right

flexure

64f

SC85

90++,80%

++,20%

−−

pT4,pN

1

4Yes

Cecum

81m

LC

5290

+++,100%

+,40%

++,60%

−pT

4a,pN0

5No

Sigma

70f

SC

120

80+++,100%

+++,80%

+++,5%

−pT

4a,pN2b

6No

Transversum

54m

LC

3170

n.a.

+++,100%

++,60%

−pT

3,pN

1,pM

1(PER)

7No

Cecum

87f

LC

105

80+++,20%

+++,90%

−−

pT3b,pN2b,pM1a

(HEP)

8No

Cecum

66f

SC

145

70n.a.

+++,90%

+++,90%

−pT

4,pN

1

9No

Ascendens

82f

SC

104

90+++,90%

++,70%

−−

pT4b,pN2b,

pM1(H

EP,PE

R)

10No

Rectum

77m

SC

8780

n.a.

+++,90%

−−

pT4b,pN1b

186 Endocr Pathol (2020) 31:182–189

a potential strong confounder for individualized therapies. It isa limitation of our study that results for all tumors with intactMMR were derived from TMA cores and it cannot becompletely ruled out that a tumor with heterogeneousMMR/MSI status has been missed among this group becausea MMR-deficient tumor area has not been represented on therespective TMA spot.

PCR analysis revealed MSI-high in all 3 cases of MMRdeficiency. Although the number of cases is too small to drawmajor conclusions, the high concordance between IHC andPCR results is not surprising. The “Bethesda Panel,” a selec-tion of 5 specific mono- and dinucleotide repeats, has beendeveloped based on data derived from colorectal cancers [16].As different microsatellite loci are not equally affected byframeshifts in the state of MSI due to an association betweentranscriptional activity and the occurrence of instability in in-dividual repeat loci [38, 39], a tissue-dependent pattern ofMSI is conceivable. Loci typically demonstrating instabilityin colorectal adenocarcinomas with MSI, i.e., the loci coveredby the “Bethesda Panel,” may be affected with comparablefrequency in colorectal NECs exhibiting MSI, just as bothtumor types certainly show genetic overlap due to their sharedorigin from large bowel mucosa.

In summary, the detection of MSI in 3 of 10 colorectalNECs but not in other neuroendocrine neoplasm suggests thatMSI affects NECs of the colorectum in a relevant manner,similar to colorectal adenocarcinomas. In contrast, MSI ishighly infrequent in NETs irrespective of tumor site. The ob-served complete homogeneity of MMR deficiency indicates

that the use of minor biopsy samples should result in highlyrepresentative data and hints towards MSI as an early event intumorigenesis. Testing for MSI early in the course of diseasemight open novel treatment options for colorectal NECs.

Acknowledgments We are grateful to Melanie Witt, Maren Eisenberg,Gabi Rieck, Sina Dietrich, and Jana Hagemann for excellent technicalassistance.

Author Contributions CF, RS, GS, WF, JS: contributed to conception,design, data collection, data analysis and manuscript writing.

WF: data analysis and manuscript writing.DH, FB, EB, TC, DP, JI: conception and design, collection of samples.CF, CHM, TA, FV, GMF, MK: collection and data analysis.WW, GS, SS: study supervision.

Funding Information Open Access funding provided by Projekt DEAL.

Compliance with Ethical Standards

Statement of Ethics The usage of archived diagnostic left-over tissuesfor manufacturing of TMAs and their analysis for research purposes aswell as patient data analysis has been approved by local laws (HmbKHG,§12,1) and by the local ethics committee (Ethics commission Hamburg,WF-049/09). All work has been carried out in compliance with theHelsinki Declaration.

Conflict of Interest The authors declare that they have no conflicts ofinterest.

Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing, adap-tation, distribution and reproduction in any medium or format, as long asyou give appropriate credit to the original author(s) and the source,

Fig. 2 Suspected MMR deficiency by TMA screening in a NET with intact MMR. For one pancreatic NET G3, TMA immunohistochemistry wasinterpreted as isolated PMS2 loss but repeated large section IHC showed intact MMR and PCR revealed microsatellite stability (MSS)

Endocr Pathol (2020) 31:182–189 187

provide a link to the Creative Commons licence, and indicate if changeswere made. The images or other third party material in this article areincluded in the article's Creative Commons licence, unless indicated oth-erwise in a credit line to the material. If material is not included in thearticle's Creative Commons licence and your intended use is not permittedby statutory regulation or exceeds the permitted use, you will need toobtain permission directly from the copyright holder. To view a copy ofthis licence, visit http://creativecommons.org/licenses/by/4.0/.

References

1. Hempelmann JA, Lockwood CM, Konnick EQ, Schweizer MT,Antonarakis ES, Lotan TL, Montgomery B, Nelson PS, KlemfussN, Salipante SJ, Pritchard CC Microsatellite instability in prostatecancer by PCR or next-generation sequencing. J ImmunotherCancer. 2018;6(1):29.

2. Sahnane N, Furlan D, Monti M, Romualdi C, Vanoli A, Vicari E,Solcia E, Capella C, Sessa F, la Rosa S Microsatellite unstablegastrointestinal neuroendocrine carcinomas: a new clinicopatholog-ic entity. Endocr Relat Cancer. 2015;22(1):35–45.

3. Stelow EB, Moskaluk CA, Mills SE. The mismatch repair proteinstatus of colorectal small cell neuroendocrine carcinomas. Am JSurg Pathol. 2006;30(11):1401–1404.

4. Olevian DC, Nikiforova MN, Chiosea S, Sun W, Bahary N, KuanSF, Pai RK Colorectal poorly differentiated neuroendocrine carci-nomas frequently exhibit BRAF mutations and are associated withpoor overall survival. Hum Pathol. 2016;49:124–134.

5. Furlan D, Sahnane N, Mazzoni M, Pastorino R, Carnevali I,Stefanoli M, Ferretti A, Chiaravalli AM, la Rosa S, Capella CDiagnostic utility of MS-MLPA in DNA methylation profiling ofadenocarcinomas and neuroendocrine carcinomas of the colon-rec-tum. Virchows Arch. 2013;462(1):47–56.

6. La Rosa S,MarandoA, FurlanD, Sahnane N, Capella C. Colorectalpoorly differentiated neuroendocrine carcinomas and mixedadenoneuroendocrine carcinomas: insights into the diagnosticimmunophenotype, assessment of methylation profile, and searchfor prognostic markers. Am J Surg Pathol. 2012;36(4):601–611.

7. House MG, Herman JG, Guo MZ, Hooker CM, Schulick RD,Cameron JL, et al. Prognostic value of hMLH1 methylation andmicrosatellite instability in pancreatic endocrine neoplasms.Surgery. 2003;134(6):902–908; discussion 9.

8. Mei M, Deng D, Liu TH, Sang XT, Lu X, Xiang HD, Zhou J, WuH, Yang Y, Chen J, Lu CM, Chen YJ Clinical implications ofmicrosatellite instability and MLH1 gene inactivation in sporadicinsulinomas. J Clin Endocrinol Metab. 2009;94(9):3448–3457.

9. Joost P, Veurink N, Holck S, Klarskov L, Bojesen A, Harbo M,et al. Heterogenous mismatch-repair status in colorectal cancer.Diagn Pathol. 2014;9:126.

10. Chapusot C, Martin L, Bouvier AM, Bonithon-Kopp C, Ecarnot-Laubriet A, Rageot D, Ponnelle T, Laurent Puig P, Faivre J, Piard FMicrosatellite instability and intratumoural heterogeneity in 100right-sided sporadic colon carcinomas. Br J Cancer. 2002;87(4):400–404.

11. Watson N, Grieu F, Morris M, Harvey J, Stewart C, Schofield L,Goldblatt J, Iacopetta BHeterogeneous staining for mismatch repairproteins during population-based prescreening for hereditarynonpolyposis colorectal cancer. J Mol Diagn. 2007;9(4):472–478.

12. Watkins JC, Nucci MR, Ritterhouse LL, Howitt BE, Sholl LM.Unusual Mismatch Repair Immunohistochemical Patterns inEndometrial Carcinoma. Am J Surg Pathol. 2016;40(7):909–916.

13. Pai RK, Plesec TP, Abdul-Karim FW, Yang B, Marquard J,Shadrach B, RomaARAbrupt loss ofMLH1 and PMS2 expression

in endometrial carcinoma: molecular andmorphologic analysis of 6cases. Am J Surg Pathol. 2015;39(7):993–999.

14. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P,Leighton S, et al. Tissue microarrays for high-throughput molecularprofiling of tumor specimens. Nat Med. 1998;4(7):844–847.

15. Tang LH, Basturk O, Sue JJ, Klimstra DS. A Practical Approach tothe Classification of WHO Grade 3 (G3) Well-differentiatedNeuroendocrine Tumor (WD-NET) and Poorly DifferentiatedNeuroendocrine Carcinoma (PD-NEC) of the Pancreas. Am JSurg Pathol. 2016;40(9):1192–1202.

16. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, EshlemanJR, Burt RW, et al. A National Cancer Institute Workshop onMicrosatellite Instability for cancer detection and familial predispo-sition: development of international criteria for the determination ofmicrosatellite instability in colorectal cancer. Cancer Res.1998;58(22):5248–5257.

17. Hinsch A, Brolund M, Hube-Magg C, Kluth M, Simon R, Moller-KoopC, et al. Immunohistochemically detected IDH1(R132H) mu-tation is rare and mostly heterogeneous in prostate cancer. World JUrol. 2018;36(6):877–882.

18. Simon R, Panussis S, Maurer R, Spichtin H, Glatz K, Tapia C,Mirlacher M, Rufle A, Torhorst J, Sauter G KIT (CD117)-positivebreast cancers are infrequent and lack KIT gene mutations. ClinCancer Res. 2004;10(1 Pt 1):178–183.

19. Pocrnich CE, Ramalingam P, Euscher ED, Malpica A.Neuroendocr ine Carcinoma of the Endometr ium: AClinicopathologic Study of 25 Cases. Am J Surg Pathol.2016;40(5):577–586.

20. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in can-cer of the proximal colon. Science. 1993;260(5109):816–819.

21. Chung HC, Lopez-Martin JA, Kao SC-H, Miller WH, Ros W, GaoB, et al. Phase 2 study of pembrolizumab in advanced small-celllung cancer (SCLC): KEYNOTE-158. J Clin Oncol .2018;36(15_suppl):8506.

22. Warth A, Korner S, Penzel R,Muley T, Dienemann H, SchirmacherP, et al. Microsatellite instability in pulmonary adenocarcinomas: acomprehensive study of 480 cases. Virchows Arch. 2016;468(3):313–319.

23. Takamochi K, Takahashi F, Suehara Y, Sato E, Kohsaka S, HayashiT, Kitano S, Uneno T, Kojima S, Takeuchi K, Mano H, Suzuki KDNAmismatch repair deficiency in surgically resected lung adeno-carcinoma: Microsatellite instability analysis using the Promegapanel. Lung Cancer. 2017;110:26–31.

24. Vanderwalde A, Spetzler D, Xiao N, Gatalica Z, Marshall J.Microsatellite instability status determined by next-generation se-quencing and compared with PD-L1 and tumor mutational burdenin 11,348 patients. Cancer Med. 2018;7(3):746–756.

25. Cortes-Ciriano I, Lee S, Park WY, Kim TM, Park PJ. A molecularportrait of microsatellite instability across multiple cancers. NatCommun. 2017;8:15180.

26. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK,et al. Mismatch repair deficiency predicts response of solid tumorsto PD-1 blockade. Science. 2017;357(6349):409–413.

27. Sorbye H, Welin S, Langer SW, Vestermark LW, Holt N, OsterlundP, Dueland S, Hofsli E, Guren MG, Ohrling K, Birkemeyer E,Thiis-Evensen E, Biagini M, Gronbaek H, Soveri LM, Olsen IH,Federspiel B, Assmus J, Janson ET, Knigge U Predictive and prog-nostic factors for treatment and survival in 305 patients with ad-vanced gastrointestinal neuroendocrine carcinoma (WHO G3): theNORDIC NEC study. Ann Oncol. 2013;24(1):152–160.

28. Mitsuhashi K, Yamamoto I, Kurihara H, Kanno S, Ito M, IgarashiH, Ishigami K, Sukawa Y, Tachibana M, Takahashi H, Tokino T,Maruyama R, Suzuki H, Imai K, Shinomura Y, Yamamoto H,Nosho K Analysis of the molecular features of rectal carcinoidtumors to identify new biomarkers that predict biological malignan-cy. Oncotarget. 2015;6(26):22114–22125.

188 Endocr Pathol (2020) 31:182–189

29. Kidd M, Eick G, Shapiro MD, Camp RL, Mane SM, Modlin IM.Microsatellite instability and gene mutations in transforminggrowth factor-beta type II receptor are absent in small bowel carci-noid tumors. Cancer. 2005;103(2):229–236.

30. Ghimenti C, Lonobile A, Campani D, Bevilacqua G, Caligo MA.Microsatellite instability and allelic losses in neuroendocrine tu-mors of the gastro-entero-pancreatic system. Int J Oncol.1999;15(2):361–366.

31. Arnold CN, Sosnowski A, Blum HE. Analysis of molecular path-ways in neuroendocrine cancers of the gastroenteropancreatic sys-tem. Ann N YAcad Sci. 2004;1014:218–219.

32. Arnold CN, Nagasaka T, Goel A, Scharf I, Grabowski P, SosnowskiA, Schmitt-Gräff A, Boland CR, Arnold R, Blum HE Molecularcharacteristics and predictors of survival in patients with malignantneuroendocrine tumors. Int J Cancer. 2008;123(7):1556–1564.

33. Arnason T, Sapp HL, Rayson D, Barnes PJ, Drewniak M, NassarBA, Huang WY Loss of expression of DNA mismatch repair pro-teins is rare in pancreatic and small intestinal neuroendocrine tu-mors. Arch Pathol Lab Med. 2011;135(12):1539–1544.

34. Sorscher S, Saroya B. A molecularly confirmed neuroendocrinetumor resulting from Lynch Syndrome. J Gastrointest Oncol.2013;4(1):95–96.

35. Serracant Barrera A, Serra Pla S, Blazquez Mana CM, Salas RC,Garcia Monforte N, Bejarano Gonzalez N, et al. Pancreatic non-functioning neuroendocrine tumor: a new entity genetically relatedto Lynch syndrome. J Gastrointest Oncol. 2017;8(5):E73-EE9.

36. Fraune C, Simon R, Hoflmayer D, Moller K, Dum D, Buscheck F,et al. High homogeneity of mismatch repair deficiency in advancedprostate cancer. Virchows Arch. 2019.

37. Fraune C, Rosebrock J, Simon R, Hube-Magg C, Makrypidi-Fraune G, Kluth M, et al. High homogeneity of MMR deficiencyin ovarian cancer. Gynecol Oncol. 2020.

38. Schuster-Bockler B, Lehner B. Chromatin organization is a majorinfluence on regional mutation rates in human cancer cells. Nature.2012;488(7412):504–507.

39. Supek F, Lehner B. Differential DNA mismatch repair underliesmutation rate variation across the human genome. Nature.2015;521(7550):81–84.

Publisher’s Note Springer Nature remains neutral with regard to jurisdic-tional claims in published maps and institutional affiliations.

Endocr Pathol (2020) 31:182–189 189